Histoplasma capsulatum (Hc) is a dimorphic fungus that is a significant cause of respiratory and systemic mycosis in normal and especially immunocompromised people. The AIDS epidemic has increased the need to understand the molecular mechanisms of pathogenesis and interactions with the host of this microorganism. Hc has respiratory route of infection, which is initiated when conidia and mycelial fragments are inhaled by mammals. Early events in pulmonary tissue, including the conversion from the infectious mold morphotype to the pathogenic yeast morphotype and entry and survival in macrophage phagolysosomes are critical events in the establishment of intracellular infection and disease. Activation of host cell (especially T cell) -mediated immunity results in containment of the infection, although latent persistent infection may occur. Hc virulence determinants have not been well-characterized, and Hc gene expression during in vivo replication and resolution of infection are not known. Much work has been done to study the biology and intracellular parasitism of Hc. Mammalian cell culture and animal models of infection have been used to examine virulence of Hc and host immune responses to this organism. Recently, molecular genetic systems have been developed that allow experimental access to Hc on a previously unexplored level. This proposal is intended to utilize the strengths of the laboratories of two investigators with distinct yet complementary approaches and skills. The Principal Investigator has developed many of the tools and techniques that enable molecular genetic manipulation of Hc. The Co-investigator has been instrumental in using mouse infection models to study immune responses to Hc in normal and immunocompromised hosts, and in identifying and examining immunogenic antigens of the organism. Delineating the importance of known genes and identifying new genes that may be expressed or repressed in vivo during acute infection and during resolution are pivotal to understanding the mechanisms by which the organism initially survives within the host's tissues, establishes infection, and causes disease. The specific aims of this proposal are: l. To apply molecular genetic approaches to determine the functional in vivo role of two prominent antigens H and M, in virulence and immunogenicity, including gene overexpression and disruption strategies and a mouse model of pulmonary histoplasmosis using both normal and CD4+ T cell-depleted animals. 2. To identify new genes, the expression of which is regulated during infection, using a novel in vivo expression technology (IVET) strategy as well as differential hybridization and differential display reverse transcriptase-polymerase chain reaction (ddRT-PCR) techniques.